The present invention relates to genetically modified yeasts in which the expression of genes responsible for cell wall synthesis are modulated.
The yeast cell wall is a dynamic organelle responsible for a number of cellular functions, the most important being physical and osmotic protection, selective permeability, cell-cell recognition and adhesion during mating and flocculation.
The cell wall of the yeast Saccharomyces cerevisiae is composed of three components, xcex2-glucan (a glucose polymer), mannoproteins and chitin (an N-acetylglucosamine polymer). The xcex2-glucan component of the cell wall consists of two polymers: a large, linear xcex2-1,3-glucan and a smaller, highly branched xcex2-1,6-glucan moiety whereas the mannoproteins are a complex of proteins modified by the attachment, via N- and O-glycosidic bonds, of mannose-containing carbohydrate chains of different length and structure.
The yeast cell wall is a very rigid, highly complex structure which determines the shape of the yeast cell and enables it to be protected from and adjusted to its ever-changing environment. A growing number of genes have been shown to participate in the biosynthesis and assembly of the major cell wall components, some of them as part of well-defined signal transduction pathways. For instance, PKC1 (the yeast homologue of protein kinase C) regulates the biosynthesis and assembly of major cell wall components by a PKC1-mediated signal transduction pathway. PKC1, in conjunction with Rho1p, regulates xcex21-3 glucan synthetase Null mutants of PKC1 can only grow in the presence of osmotic stabilisers. Loss of PKC1 function results in a cell-cycle-specific osmotic stability defect.
A further gene relevant to the cell wall is the recently identified SRB1 (also known as PSA1 or VIG9) yeast gene which is essential for growth and encodes GDP-mannose pyrophosphorylase, an enzyme responsible for the production of a major substrate for all kinds of mannosylation reactions, including the biosynthesis of cell wall mannoprotein. A SRB1/PSA1 null mutation is lethal whereas a decrease in SRB1/PSA1 function (by inhibiting the expression of SRB1/PSA1) leads to defects in bud growth, bud site selection, and cell separation, in addition to increases in cell permeability and cell lysis.
Another gene which regulates the cell wall is PDE2 (the gene encoding a high-affinity cAMP phosphodiesterase) which is part of the RAS/cAMP dependent pathway in yeast. We recently identified PDE2 as a multi-copy suppressor of srb1-1 (a mutation which depends on sorbitol for growth). Moreover, strains carrying a pde2 deletion share a number of phenotypes with srb1-1 mutants, including lysis upon osmotic shock.
The yeast cell wall influences the characteristics of the yeast cell in a number of ways and it is desirable to modulate the wall to confer desirable properties on yeast.
For instance, the yeast cell wall acts as a barrier which can obstruct the liberation of protein expressed within yeast cells. This effect is of particular significance to the biotechnology industry which uses yeasts for protein production and for the production of recombinant proteins in particular. For some proteins, it is possible to use the yeast secretion pathway to release the protein from the cell, thus obviating the need for mechanical or enzymic degradation of the cell wall. However, many proteins cannot be exported by the secretion pathway and are retained within the cell in a membrane-associated form. Some protein complexes, such as virus-like particles (VLPs) are also impossible to recover by the secretion route. It may be seen, therefore, that the rigid cell wall of yeast is a major barrier to the efficient operation of downstream processes leading to protein isolation and purification. In the food industry, yeast cell extracts are used as dietary supplements and flavourings, again requiring efficient methods of cell lysis that do not compromise the nutritional or organoleptic properties of the yeast cell extract.
The use of lysis mutants represent an alternative to mechanical/chemical disruption for the efficient recovery of yeast cell contents. Some attempts have been made to develop such mutants. For instance, WO 92/01798 concerns the use of a srb1-1 mutant. However this specification related to a DNA sequence deposited under the Budapest treaty which was first believed to code for the SRB1 gene but has since been shown to be the PDE2 gene. WO 96/02629 also relates to a lysis mutant and concerns the use of a mpk1/slt2 mutant which is stated to release intracellular proteins, including VLPs, following a temperature shift and osmotic shock. However, the application of both types of lytic mutants has significant drawbacks. The osmotic stabiliser required to enable the growth of a srb1-1 mutant is either expensive (sorbitol) or corrosive to the fermentor (NaCl) thus precluding the use of this mutant nil in large-scale processes. The temperature shift involved in the use of the mpk1/slt2 mutant requires considerable energy input and can also trigger the degradation or re-modelling of the proteins released from the lysed cells.
It is therefore an object of the present invention to provide yeast mutants which obviate or mitigate the abovementioned disadvantages.
According to a first aspect of the invention, there is provided a yeast cell containing the SRB1/PSA1 gene and the PKC1 gene or functional derivatives thereof each operatively linked to a heterologous inducible promoter.
We have found that yeast cells containing the SRB1/PSA1 gene and the PKC1 gene or functional derivatives thereof each operatively linked to an inducible promoter (e.g. the cells according to the first aspect of the invention) may be used in applications where the induction of cell lysis is desirable. For instance, induction of yeast cell lysis is useful for isolating protein expressed within a yeast cell which is not readily secreted into the medium in which the cells are growing. Thus according to a second aspect of the present invention, there is provided a method of regulating yeast cell lysis comprising:
(i) growing yeast cells containing the SRB1/PSA1 gene and the PKC1 gene or functional derivatives thereof each operatively linked to an inducible promoter in a growth medium which activates the inducible promoter such that SRB1/PSA1 and PKC1 are expressed from said cells; and
(ii) when lysis is required, growing the cells in a modified growth medium which represses SRB1/PSA1 and PKC1 expression such that cell lysis is induced.
The present invention is based upon our efforts to develop conditional lysis mutants that do not require special media in which to grow (e.g. sorbitol supplemented) or temperature shifts. We placed various genes that have been shown to contribute, in different ways, to cellular integrity of S. cerevisiae under the control of inducible promoters and examined A whether or not repression of the gene (by altering the composition of the media in which the yeast is grown such that the promoter is inactivated) modulates cell lysis. We found that repression of some of the genes tested, for instance PDE2, did not significantly influence lysis. These genes were therefore unsuitable candidates to be modulated to generate an improved lytic yeast strain.
We found that repression of the SRB1/PSA1 gene in yeast cells grown in normal media resulted in a reduction in cell growth, cells gradually losing their viability and integrity and the release of about 7% of total protein into the medium 24 hours after repression was induced. Furthermore we found that repression of PKC1 led to more extensive release of cellular protein into the medium (approximately 18% of total protein into the medium 24 hours after repression was induced) although cell growth was not significantly altered. These results were expected because, although it is known that yeast cells carrying the srb1-1 allele can grow in osmotically-buffered media, such mutanits lyse upon hypoosmotic shock. It is also known that cells bearing the pkc1 mutation can grow in osmotically-bufferred media (e.g. in the presence of 10% (w/v) sorbitol) but quickly lyse upon osmotic shock.
We also repressed expression of both SRB1/PSA1 and PKC1 in yeast cells and found that under these circumstances the yeast cultures underwent substantial lysis which permitted the efficient release of both homologous and heterologous proteins from the yeast. In fact, as illustrated in Example 1, lysis was surprisingly more extensive than observed for cells in which SRB1/PSA1 and PKC1 were repressed singularly (about 30% of total protein was released into the medium 24 hours after repression was induced) which shows that the lysis phenotype conferred by the repression of SRB1/PSA1 and PKC1 was additive. Thus cells according to the first aspect of the invention are of particular utility as it is possible to grow the cells without significant lysis and then at a predetermined time during the fermentation induce extensive lysis of the yeast cells.
Cells according to the first aspect of the invention may be formed from yeast strains with normal SRB1/PSA1 and PKC1 expression. Preferably the yeast is Saccharomyces cerevisiae or strains thereof. Examples of such yeast strains include ZO123 and FY23. The conservation of gene function in different yeast species means that other types of yeasts (particularly those that are currently exploited for heterologous gene expression such as Pichia pastoris, Hansenula polymorpha and Kluyveromyces lactis) may be used to form cells according to the first aspect of the invention in which their SRB1/PSA1 and PKC1 homologues are operatively linked to an inducible promoter.
The endogenous promoters of the SRB1/PSA1 gene and the PKC1 gene are not readily inducible in such strains and it is therefore necessary to modify genetically yeast such that SRB1/PSA1 and PKC1 expression from the yeasts is inducible. This may be achieved in a number of ways. For instance, yeast cells may be transformed with DNA molecule(s) comprising an inducible promoter(s) such that the inducible promoter(s) take over control of transcription of the endogenous SRB1/PSA1 and PKC1 genes. These DNA molecules are preferably designed such that they will integrate into the yeast genome and replace the region of DNA containing the endogenous SRB1/PSA1 and PKC1 promoters (as appropriate). As a result the inducible promoter introduced into the cell becomes operatively linked to the SRB1/PSA1 and PKC1 genes and can control their expression. Alternatively the cells may be transformed with a first recombinant DNA molecule comprising an inducible promoter operatively linked to the SRB1/PSA1 gene and/or a second recombinant DNA molecule comprising an inducible promoter operatively linked to the PKC1 gene. These recombinant DNA molecules are designed such that they will integrate and replace by homologous recombination the endogenous SRB1/PSA1 and PKC1 genes respectively. The DNA molecules and recombinant DNA molecules used for transforming yeast cells are preferably incorporated in a suitable vector which bears a DNA sequence which allows homologous recombination between the vector and the DNA at the site of the endogenous promoter/gene.
The cells according to the first aspect of the invention may also be derived from yeasts which are srb1-1 and/or pkc mutants. These mutants have a lytic phenotype and are only able to survive when grown in osmotically buffered media. However we have found that these cells may be transformed with an expression cassette comprising an inducible promoter and DNA sequences encoding suitable genes to replace the mutated gene to form cells according to the first aspect of the invention which display a normal phenotype (i.e. they are not osmotically sensitive or liable to lyse spontaneously) in permissive growth media conditions (which allows activation of the inducible promoter) but will lyse when the media is modified such that gene expression is repressed. Yeast cells to be modified may be transformed with the abovementioned recombinant DNA molecules (or vectors bearing such molecules) to form cells according to the first aspect of the invention in which the recombinant DNA molecules either integrate into the genome of the mutant yeast or which may subsist (and ideally autonomously replicate) in the cytosol of the yeast cell. Examples of srb1-1 and/or pkc mutant cells which may be used include the ZO124 strain of Saccharomyces cerevisiae. 
The SRB1/PSA1 gene and the PKC1gene (or functional derivatives thereof) may each be operatively linked to a number of inducible promoters. The inducible promoter may, for example, be the GAL1 promoter (inducible by galactose) or the TET promoter (inducible by tetracyclin).
Preferred promoters are ones which may be regulated by an agent contained within the media within which the cells are grown. Such agents are ideally readily available, inexpensive, soluble in normal yeast growth medium and do not adversely effect proteins released from lysed cells. We have found that the methionine regulated promoter, pMET3 (Mountain et al., 1991 Yeast 7: 781-803) fulfils these criteria as its modulator (methionine) may be easily included or excluded in growth medium as required. Thus pMET3 is a preferred promoter.
The pMET3 promoter drives gene expression in the absence of methionine. Therefore in methionine-free media SRB1/PSA1 and PKC1 expression occurs. However when the media is modified by the addition of methionine, gene expression is repressed and cell Iysis induced.
DNA sequences corresponding to pMET3 may be used as a DNA molecule for transforming yeast cells although it is preferred that pMET3 is contained within a vector (i.e. as part of a larger DNA molecule containing other functional elements). A preferred vector, named pRS316-pMET3, comprises a pRS316-based plasmid (described in Sikorski and Heiter (1989) Genetics 122: 19-27) which contains the MET3 promoter (Yeast cenome Accession no. X06413). The construction of pRS316-pMET3 is described in detail in Example 1.
pRS316-pMET3 may be used to form recombinant vectors which contain preferred recombinant DNA molecules pMET3-PKC1 and pMET3-SRB1.
Preferred derivatives of pRS316-pMET3 which contain pMET3-PKC1 include pRS316-pMET3-PKC1 is described in detail in Example 1. Other preferred derivatives of pRS316-pMET3 are designed to allow integration of the pMET3-PKC1 regulation cassette at the homologous PKC1 locus. For instance, pRS316-F1F2-pMET3-PKC1 is constructed by inserting a PKC1 upstream flanking region (which was designated F1F2 and has the nucleotide sequence listed below) between the KpnI and SphI sites of pRS316-pMET3-PKC1.
Nucleotide sequence of the F1F2 DNA fragment:
ACAAGCAGCTGATGAAAAGCCAAGACATAAGTATTGT TGCCCACACT GTGGGTCTTCATTTCCAAGATGTGCCATATGTCTCATGCCTCTAGGAA CGTCAAACTTACCTTTTGTAATAAATGGGACGCAATCACGCGATCAAT GCAGACAGAAGACTCTCAAGATGGTGCAAATCGCGAACTCGTAAGTA GAAAACTGAAGTTGAACGAGTGGTTCAGCTTCCTGTTTGAGTTGCAACCA TGGTATGCATGCCGGTCACGCTGAAGAATGGTTTGACAGACATAATGTT TGTCCCACTCCAGGTT (SEQ. I.D. NO.14)
pRS316-F1F2-pMET3-PKC1 may also be further modified to introduce the TRP1 gene as a selectable marker (a DNA molecule corresponding to Yeast genome accession No. V01341 or J01374) between the SphI and SalI sites to form the construct pRS316-F1F2-TRP1-pMET3-PKC1. pRS316-F1F2-TRP1-pMET3-PKC1 is particularly useful when forming cells according to the first aspect of the invention because it may be digested with KpnI and SacI and the fragment containing F1F2-TRP1-pMET3-PKC1 used to transform a host yeast strain.
SRB1.9e is a preferred recombinant vector which contains the recombinant DNA molecule pMET3SRB1 and is described in more detail in Example 1. Plasmid pSRB1-9e is particularly useful when forming cells according to the first aspect of the invention because it may be digested with ApaI and BstI107I and the pMET3-SRB1-LEU2 fragment obtained in this way used to transform a yeast cell.
Preferred cells according to the first aspect of the invention may be formed using the above described constructs. For instance, yeast strain ZO-123 (MATa his3 leu2 trp1 ura3) which expresses both SRB1/PSA1 and PKC1 may be modified such that the transcription of the endogenous SRB1/PSA1 and PKC1 genes are brought under the control of pMET3 (e.g. by integrating pMEFT3 into the yeast genome such that it replaces the endogenous SRB1 and PKC1 promoters). Alternatively pRS316-F1 F2-pMET3-PKC1 or pRS316-F1F2-TRP1-pMET3-PKC1 in conjunction with SRB1.9e may be used to integrate the pMET3-PKC1 and pMET3-SRB1 cassettes respectively into ZO-123 to form ZO-127 which is a particularly preferred cell according to the first aspect of the invention.
We have found that lysis may be regulated according to the method of the second aspect of the invention by growing yeast cells according to the first aspect of the invention in a growth medium which activates the inducible promoter such that SRB1/PSA1 and PKC1 are expressed from said cells. Then, after a predetermined time, the cells may be switched to growth in the modified growth medium such that SRB1/PSA1 and PKC1 expression is repressed and cell lysis induced.
The manner in which SRB1/PSA1 and PKC1 gene expression is regulated according to the method of the second aspect of the invention will depend upon which inducible promoter is being used. This regulation is dependent upon the exact concentration of an agent capable of modulating promoter activity contained within the growth medium. We have found that addition to the media of methionine to a concentration of between 0.05 mM and 20 mM will inhibit expression of SRB1/PSA1 and PKC1 from cells transformed with pMET3-SRB1 and pMET3-PKC and thereby induce lysis whereas the same cells grown in the absence (or minimal concentration) of methionine are able to grow unimpeded. Preferably a concentration of between about 0.05 mM and 5 mM methionine in the media and most preferably a concentration of about 2 mM methionine in the media is used to induce lysis.
The growth medium used according to the method of the second aspect of the invention should be readily adaptable such that it may be in either of two forms: one which permits activation of the inducible promoter and thereby SRB1/PSA1 and PKC1 expression; and a second form which is modified such that SRB1/PSA1 and PKC1 gene expression is repressed. This repression may be effected by removal of an agent which activates the promoter but is preferably effected by addition to the media of an agent which inhibits the promoter.
The growth medium should contain sufficient amounts of nutrients (i.e carbohydrate, nitrogen source etc) required to allow optimal, growth of yeast when SRB1/PSA1 and PKC1 are not being repressed.
The exact composition of the medium depends upon a number of factors (for instance the specific yeast used). Purely by way of example a suitable growth medium is F1 medium which comprises:
Mineral Salts Final Concentration in F1-Medium
Trace Elements
Vitamins
+ carbohydrate substrate
+/xe2x88x92 agent which modulates the inducible promoter
The composition of the media and the modified form thereof ideally only differ by the inclusion or exclusion of an agent which modulates the inducible promoter. The type of agent used will depend upon which specific promoter is used. When cells are used in which SRB1/PSA1 and PKC1 are operatively linked to the pMET promoter, the media permissive for yeast cell growth should be free of methionine. Methionine may be added to the medium as required to form the modified media in which lysis is induced.
The method of the second aspect of the invention may be readily adapted for the purposes of isolating protein from yeast cells. Once yeast cells have been lysed yeast cell debris/hosts may easily be separated from the protein released from the cells (e.g. by filtration, sedimentation and/or centrifugation). The protein may then be further purified using conventional biochemical techniques. The method is most suitable for isolating recombinant proteins expressed from genetically engineered yeast cells.
Another characteristic of yeast which is determined by the cell wall is its ability to flocculate. Unlike adhesion in mating, which is induced by highly specific pheromones, flocculation is an asexual aggregation of cells which is a very useful characteristic in industrial yeast strains. Flocculation is exploited in fermentations such as beer-brewing, wine-making, and fuel ethanol production because it leads to efficient separation of cells from the fermentation liquor.
Two types of flocculation phenotypes have been described. The FLO1 type, caused by FLO1/FLO5/FLO8, is Ca2+-dependent and inhibited by mannopyranoses. The NewFlo phenotype, on the other hand, is prevented by both manno- and glucopyranoses. The FLO1 gene, which is located on chromosome 1, has been reported to encode a GPI-anchored, cell surface protein with its amino terminus exposed to the medium. FLO5 is highly homologous to FLO1 and is also found on chromosome 1. FLO8, previously mapped to chromosome 1 and said to be allelic to FLO1 has recently been reassigned to chromosome V and demonstrated to mediate flocculation via transcriptional activation of FLO1. More recently, a new flocculation gene, named FLO2, has been cloned and localised to chromosome XII; its function remains unclear, although it can complement flo1 mutations. Other genes, like TUP1 and SNN6, also act on yeast cell flocculation via transcriptional regulation.
Flocculation in S. cerevisiae is thought to be a result of interactions between lectin-like cell surface proteins (termed flocculins), encoded by the FLO genes. and the cell wall mannan. This hypothesis is supported by the following findings: loss of flocculation capacity following protease treatment, efficient dispersion of flocs by mannose and its derivatives and the failure of certain mnn mutant cells to co-flocculate with flocculant cells. So far, studies of flocculation have centred on the cloning and characterisation of dominant flocculation genes and the elucidation of their transcriptional regulation. Less attention has been paid to the effect of changes of cell wall structure on flocculation.
We have found that cells in which the PKC1 and/or SRB1/PSA1 gene or functional derivatives thereof are operatively linked to heterologous promoters may be used in applications in which the induction of flocculation is desirable (e.g. fermentation reactions such as for the production of alcohol).
According to a third aspect of the present invention, there is provided a method of regulating yeast cell flocculation comprising:
(i) growing yeast cells containing the PKC1 gene or functional derivatives thereof operatively linked to an inducible promoter in a growth medium which activates the inducible promoter such that PKC1 is expressed; and
(ii) when flocculation is required, growing the cells in a modified growth medium which represses PKC1 expression such that flocculation is induced.
According to the third aspect of the invention we have found that the repression of PKC1 expression makes it possible to induce flocculation. Therefore cells containing the PKC1 gene or functional derivatives thereof operatively linked to a heterologous inducible promoter (such as pMET3) are useful when it is desired to induce flocculation. Such cells are particularly useful when the simultaneous induction of lysis and flocculation is required (as PKC1 repression also causes lysis). The induction of lysis and flocculation is desirable when purifying proteins released from yeasts. The induced lysis liberates the contents of the cell whereas the induced flocculation will favour sedimentation of the cell ghosts/debris and thereby separate cell contents (which will remain in the media) from the cell ghosts/debris.
Cells suitable for use according to the method of the third aspect of the invention include cells in which only PKC1 is under the regulation of an inducible promoter and include:
(i) ZO124 transformed with pRS316-pMET3-PKC1, pRS316-F1F2-pMET3-PKC1 or pRS316-F1F2-TRP1-pMET3-PKC1 (see Example 1);
(ii) ZO123 transformed with pRS316-pMET3-PKC1 or pMET3-PKC1 containing fragments derived from pRS316-F1F2-pMET3-PKC1 or pRS316-F1F2-TRP1-pMET3-PKC1 (see Example 1); and
(iii) yeast strain ZO-126 (see Example 2).
We have also found that cells may be developed which have a flocculating phenotype by removing SRB1/PSA1 from under the control of its endogenous promoter and placing the gene under the control of a heterologous promoter (which may be inducible or constitutive). Such cells flocculate but do not lyse to a significant extent and are therefore useful in industrial applications where flocculation is of primary importance (e.g. for sedimenting yeast during the brewing process). Thus according to a fourth aspect of the invention there is provided a method of fermentation comprising growing yeast cells containing the SRB1/PSA1 gene or functional derivatives thereof operatively linked to a heterologous promoter in a growth medium in which SRB1/PSA1 expression is regulated by the heterologous promoter whereby said cells flocculate.
SRB1/PSA1 expression may be regulated in cells used according to the fourth aspect of the invention by an inducible promoter or a constitutive promoter. pMET3 is a preferred promoter for regulating SRB1/PSA1 expression in cells used according to the fourth aspect of the invention.
Examples of cells in which SRB1/PSA1 is under the regulation of an inducible promoter include:
(i) ZO125 (ZO 123 cells transformed with pMET3-SRB1); and
(ii) FY23SRB1MET3.
Although we do not wish to be bound by any hypothesis we believe that the flocculation phenotype caused when SRB1/PSA1 is transcribed from pMET3 is not due to the gene""s underexpression but, rather, is the result of its constitutive expression. Cell viability is not affected by the constitutive expression of SRB1/PSA1, suggesting that sufficient Srb1/Psa1p is synthesised under the control of pMET3 to allow yeast to go through its cell cycle. However, when SRB1/PSA1 is expressed from its own promoter, its transcription level increases some 4- to 6-fold at START. Thus the constitutive expression of SRB1/PSA1 from pMET3 could hyperactivate glycosylation at all other cell cycle phase which may lead to enhanced cell growth and flocculation.
According to a fifth aspect of the invention there is provided a method of fermentation comprising growing yeast cells containing the SRB1/PSA1 and PKC1 gene or functional derivatives thereof operatively linked to a heterologous promoter in a growth medium in which SRB1/PSA1 and PKC1 expression is regulated by the heterologous promoter whereby said cells flocculate.
We believe cells used according to the method of the fifth aspect of the invention have a flocculating phenotype because SRB1/PSA1 is not regulated by its endogenous promoter in such cells. These cells may comprise:
(i) PKC1 operatively linked to an inducible promoter and SRB1/PSA1 linked to any heterologous promoter; or
(ii) both PKC1 and SRB1/PSA1 operatively linked to an inducible promoter (i.e. cells according to the first aspect of the invention).
The method of the fifth aspect of the invention may be used when it is desirable to induce lysis (e.g. according to the method of the second aspect of the invention) at a predetermined time during the fermentation as well as flocculation. For instance, this may be achieved by adding methionine (0.05 mM-20 mM) to the growth medium when PKC1 is operatively linked to a methionine regulated promoter such as pMET3.
According to a sixth aspect of the invention, there is provided a yeast cell containing the PKC1 gene or functional derivatives thereof operatively linked to a heterologous inducible promoter.
Cells according to the sixth aspect of the invention may be employed in the method according to the third aspect of the invention. Such cells may contain the PKC1 gene or functional derivatives thereof operatively linked to any inducible promoter described above for use in cells according to the first aspect of the invention.
Preferred cells according to the sixth aspect of the invention include:
(i) ZO124 transformed with pRS316-pMET3-PKC1, pRS316F1F2-pMET3-PKC1 or pRS316-F1F2-TRP1-pMET3-PKC1 (see Example l);
(ii) ZO123 transformed with pRS316-pMET3-PKC1 or pMET3-PKC1 containing fragments derived from pRS316-F1F2-pMET3-PKC1 or pRS316-F1F2-TRP1-pMET3-PKC1 (see Example 1); and
(iii) yeast strain ZO-126 (see Example 2).
According to a seventh aspect of the invention, there is provided a yeast cell containing the SRB1/PSA1 gene or functional derivatives thereof operatively linked to a heterologous promoter.
Cells according to the seventh aspect of the invention may be employed in the method according to the fourth aspect of the invention. Such cells may contain the SRB1/PSA1 gene or A functional derivatives thereof operatively linked to any heterologous promoter (including inducible promoters). Preferred promoters are described above for use in cells according to the first aspect of the invention.
Examples of cells according to the seventh aspect of the invention include:
(i) ZO125 (ZO 123 cells transformed with pMET3-SRB1); and
(ii) FY23SRB1MET3.
According to a eighth aspect of the invention, there is provided a yeast cell containing the PKC1 gene or a functional derivative thereof operatively linked to a heterologous inducible promoter and the SRB1/PSA1 gene or a functional derivative thereof operatively linked to a heterologous promoter.
Cells according to the eighth aspect of the invention may be employed according to the method of the fifth aspect of the invention. Such cells may be the same as cells according to the first aspect of the invention except the SRB1/PSA1 gene or a functional derivative thereof may be operatively linked to any heterologous promoter.
Optimal growth of yeasts used according to either the methods of the second or third aspects of the invention can be dependent upon the fermenter in which the yeast are grown. Fermenters will usually comprise one or more of:
1. Rotors or similar devices for agitating the yeast culture.
2. An air (or oxygen) supply.
3. An inlet for addition of nutrients or agents which modify the medium.
4. A means of extracting waste products and/or proteins produced
5. A thermostat and means of regulating temperature.
Preferred fermenters are those already known to the art for the culture of yeast. The type of fermenter used will depend upon whether the yeast cells are being grown in the laboratory by potage, as a pilot plant or in full industrial scale-up (e.g. for industrial production of yeast proteins).
When cells are cultured in the modified growth medium (which represses SRB1/PSA1 and PKC1 expression such that lysis is induced) according to the second aspect of the invention, the culture conditions do not need be as stringently regulated as during the growth phase because cell viability is not relevant when lysis is induced. However it will be appreciated that the media should not be allowed to change (e.g. undesirable pH or temperature changes) such that the liberated yeast cell contents (e.g. a recombinant protein) are denatured or corrupted.
The culture conditions required for cells grown in modified growth medium according to the third aspect of the invention will depend upon whether flocculation only (whilst maintaining cell viability) or whether flocculation and lysis is desired. If it is desired to maintain viability similar culture conditions as used for growth in the permissive media should be maintained whereas if lysis is to be induced the comments of the preceding paragraph apply.